SYTTY
DEVELOPMENT OF MOLECULAR METHODS FOR DETECTION OF HARMFUL MICRO-ORGANISM IN WORK ENVIRONMENT: FUNGI
Project leader: Merja Suutari, National Public Health Institute,
P.O.Box 95, FIN-70701 Kuopio, Finland, tel. +358-17-201 159, e-mail: Merja.Suutari@ktl.fi
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Financing SYTTY organization: The Finnish Work Environment Fund
EXTENDED ABSTRACT
In the work environment, remarkable amount of Finnish citizens are exposed to bioaerosols containing microbes or their biosynthesis products, such as pigments, antibiotics, cell wall components, spores, and lytic enzymes. These bioaerosols cause several illnesses related to the respiratory tract, part of them may be irritative and less serious symptoms, but they can also lead to serious chronic diseases. Bioaerosols are especially related with the presence of water leakage and fumic organic materials in buildings. In practical work hygiene, the fast detection of problems related to especially harmful micro-organisms is important. Traditional cultivation methods do not always reveal the most harmful microbes, and they are relatively slowly. More sensitive and fast methods are needed for the work hygiene control and design of healthy work environment. The aim of this project was to develop PCR-based, genetic methods for the detection and identification of the most important fungi in work environment, and use methods for studies on work environment bioaerosols. The fungi chosen to the study were Stachybotrys atra, Fusarium spp., Trichoderma spp. and Aspergillus versicolor.
The molecular methods for detection of fungi were screened from the literature, and tests for Stachybotrys chartarum (Stachybotrys atra), Fusarium spp. and Aspergillus spp. were found. Because of lack of method specific for Aspergillus versicolor, rRNA gene sequences of three strains were determined, in order to separate them from the other Aspergillus spp. by e.g. restriction enzyme digestion or fluorescence probes. No tests for Trichoderma spp. were found, although some of their ribosomal RNA gene sequences exists in gene libraries, and probes specific for fungi in general exist. These tests were not used in this project for the analyses of work environment samples. In this and previous project (The Finnish Work Environment Fund, proj. no 96028 and 98103), two primer pairs were developed for the detection of streptomycetes 16S rRNA gene sequences from environmental samples. The primers were used for PCR amplification template DNA isolated from two separate soil samples and building materials. The fragments produced were cloned, and at least 40 clones were sequenced per primer pair without false positive results. The tests recover over 95 % of streptomycetes sequences present in EMBL and GenBank data libraries.
In studies on biological problems caused by microbes in work environment Streptomycetes were used instead of fungi, because during the PCR-tests development phylogenetic information was obtained on their prevalence in buildings in relation to the natural environment. The combining this diversity information with specific nutritional and pH-demands, and toxicity at different growth conditions enabled the evaluation of key biological risks in work environment related to streptomycetes. The Streptomyces spp. present in buildings have nearly the same phylogenetic profile as those in the natural forest soil. The detection of streptomycetes from buildings by cultivating on tryptone-yeast extract-glucose (THG) medium selected lightly so that not all Streptomyces coelicolor –like strains were detected. However, in general THG-medium was one of the best for screening of streptomycetes from e.g. the air of work environments.
New information has been obtained on factors supporting the microbial
growth and harmful compound biosyntesis in work environment. In the presence
of water leakage streptomyctes normally present in natural environment
apparently begin to grow in buildings. Strepmycetes are able to degrade
complex organic compounds (starch, casein, pectin) over a broad growth
pH range. They secret metabolites harmful to humans and inhibit e.g, the
fungal growth. The strains isolated from water-damaged buildings also sporulated
well. The biological responses induced by streptomycetes spores in cell
cultures varied depending on growth and sporulation conditions.